image_2.PDF

<p>Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.</p

image_3.PDF

<p>Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.</p

image_1.PDF

<p>Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.</p

table_2.docx

<p>Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.</p

table_1.docx

<p>Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.</p

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells <i>In Vitro</i> than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

<div><p>Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells <i>in vitro</i>. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 <i>in vitro</i> and <i>in vivo</i> as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells <i>in vitro</i> results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells <i>in vitro</i> for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.</p></div

Killing of K562 <i>in vivo</i> by NK cells from CBCD34⁺ and PBCD34⁺ cultures.

<p>NSG mice were injected with GFP-K562 cells followed by CBCD34⁺-NK cells or PBCD34⁺-NK cells 24 h later. (<b>A</b>) Percentage of GFP-K562 cells detected in the BM, liver, lungs and spleen of NSG mice. (<b>B</b>) Percentage of NK cells detected in the BM, liver, lungs and spleen of NSG mice. The statistical analysis was performed using Mann-Whitney test. * <i>P</i><0.05.</p

NK cell development in CBCD34⁺ and PBCD34⁺ cultures.

<p>NK cell stages 1–4 for one representative sample from CBCD34⁺ (n = 8) (<b>A</b>) and PBCD34⁺ (n = 6) (<b>B</b>) cultures. Percentages are gated from CD3⁻ cells according to the following NK cell stages: stage 1: CD34⁺CD117⁻CD94⁻, stage 2: CD34⁺CD117⁺CD94⁻, stage 3: CD34⁻CD117⁺CD94⁻ and stage 4: CD34⁻CD117^+/−CD94⁺. (<b>C</b>) NK cell stages are shown for one representative sample for CBCD34⁺ (n = 8, upper panel) and PBCD34⁺ (n = 6, bottom panel) cultures at different time points. Stage 1 and 2 are from the CD3⁻CD94⁻ gate and stages 3 and 4 from the CD3⁻CD34⁻ gate. Transcriptional analysis for each time point is shown for transcription factors involved in NK cell differentiation (left panel) and maturation (right panel) for CBCD34⁺ (n = 4) (<b>D</b>) and PBCD34⁺ (n = 3) (<b>E</b>) cultures. Values are normalized using three reference genes. Higher ratio values correspond to less mRNA expression.</p

Effects of IL-12 stimulation on the function of the differentiated NK cells.

<p>NK cells were incubated with IL-12 for 4 h, 24 h or 40 h. (<b>A</b>) The figure illustrates the effect of IL-12 on the secretion of IFN-γ and (<b>B</b>) TNF-α measured by ELISA after incubation with PMA&Iono. (<b>C</b>) The graph depicts the intracellular expression of IFN-γ after incubation with PMA&Iono. (<b>D</b>) The graph shows CD107a degranulation after incubation with PMA&Iono. (<b>E</b>) NK cell killing capacity against ⁵¹Cr labeled K562 cells or (<b>F</b>) P815 cells coated with anti-CD16. The effector-target ratio used was 10∶1. Statistical analysis was performed using Mann-Whitney test. * <i>P</i><0.05, ** p<0.005.</p

NK cell production from CBCD34⁺ and PBCD34⁺.

<p>(<b>A</b>) Total fold expansion and (<b>B</b>) CD3⁻CD56⁺ NK cell number of CBCD34⁺ (n = 8) and PBCD34⁺ (n = 6) cultures at different time points. (<b>C</b>) Representative plot of CD56 <i>vs</i> CD16 from the lymphocyte gate for CBCD34⁺ and PBCD34⁺ cultures. (<b>D</b>) Representative side scatter <i>vs</i> CD56⁺ plots for CBCD34⁺ (upper row) and PBCD34⁺ (bottom row) cultures at days 7, 14, 21, 28, 35 and 42. Mann-Whitney test was performed, * <i>P</i><0.05, ** <i>P<</i>0.005.</p